An Integrated Human Mammary Epithelial Cell Culture System for Studying Carcinogenesis and Aging

Abstract

Experimental examination of the agents and processes that may propel or prevent human breast carcinogenesis can be facilitated by in vitro model systems of transformation, starting with normal cells, that accurately reflect the in vivo biology. Model systems that can replicate the types of alterations seen during in vivo progression offer the potential to understand the mechanisms underlying progression and to examine possible means of individualized prevention and treatment. To this end, we have developed an experimentally tractable human mammary epithelial cell (HMEC) culture system that has been used to examine the normal processes governing HMEC growth, differentiation, aging, and senescence and how these normal processes are altered during immortal and malignant transformation. Isogenic cells at different stages of multistep carcinogenesis were generated by exposing normal finite lifespan HMEC to a variety of oncogenic agents that may play an etiologic role in breast cancer. Examination of the molecular alterations present at each stage has indicated that this model is consistent with observed multistep carcinogenesis in vivo. We have seen that varying target cell type, and oncogenic agents used, can lead to multiple distinct molecular pathways of transformation, although the full diversity of human breast cancer cell types has not yet been generated in culture models. Using this integrated system, we have formulated a comprehensive model of the proliferative barriers normal HMEC must overcome to gain immortality and malignancy. Our data provide insights on acquisition of cancer-associated properties and suggest that the most crucial step in breast cancer progression involves the transition from a finite to an indefinite lifespan. For example, we see that genomic instability originates in finite lifespan HMEC when telomeres become critically short and engage in telomeric associations and is then maintained in resultant immortalized and malignant lines. Direct genomic targeting of the tumor-suppressive senescence barriers can produce lines lacking gross genomic errors, supporting the hypothesis that genomic instability is a mechanism to generate cancer-causing errors, but is not necessary per se. Immortalization through telomerase reactivation was also associated with acquisition of resistance to TGFβ growth inhibition and to oncogene-induced senescence (OIS) and with large-scale changes in gene expression and epigenetic marks. Being able to examine the progressive changes that fuel malignancy, starting with normal cells, provides an integrated perspective that can reveal novel information on the origins and consequences of individual cancer-associated aberrations.